Edited by Wolfgang J. Miller Pierre Capy Mobile Genetic Elements Protocols and Genomic Applications Volume 260 METHODS IN MOLECULAR BIOLOGY TM METHODS IN MOLECULAR BIOLOGY TM Edited by Wolfgang J. Miller Pierre Capy Mobile Genetic Elements Protocols and Genomic Applications TEs as Natural Molecular Tools 1 1 From: Methods in Molecular Biology, vol. 260: Mobile Genetic Elements Edited by: W. J. Miller and P. Capy © Humana Press Inc., Totowa, NJ 1 Mobile Genetic Elements as Natural Tools for Genome Evolution Wolfgang J. Miller and Pierre Capy Summary Transposable elements (TEs) are ubiquitous components of all living organisms, and in the course of their coexistence with their respective host genomes, these parasitic DNAs have played important roles in the evolution of complex genetic networks. The interaction between mobile DNAs and their host genomes are quite diverse, ranging from modifications of gene structure and regulation to alterations in general genome architecture. Thus over evolutionary time these elements can be regarded as natural molecular tools in shaping the organization, structure, and function of eukaryotic genes and genomes. Based on their intrinsic properties and features, mobile DNAs are widely applied at present as a technical “toolbox,” essential for studying a diverse spectrum of biological questions. In this chapter we aim to review both the evolutionary impact of TEs on genome evolution and their valuable and diverse methodologi- cal applications as the molecular tools presented in this book. Key Words: Transposable elements; selfish DNAs; genome evolution; neogene formation; heterochromatin; stress induction; molecular tools. 1. Introduction Many organisms contain far more repetitive DNA sequences than single- copy sequences. Repetitive sequences include mobile genetic DNAs that are universal components of all living genomes. Transposable elements (TEs) are gene-sized segments of DNA with the special ability to move between differ- ent chromosomal locations in their hosts’ genome. Today the genomes of vir- tually all eukaryotic and prokaryotic species are known to contain significant numbers of TEs. 1.1. Occurrence and Classification In some bacterial species, up to 10% of the genome is composed of insertion sequences (IS elements), while in eukaryotes these elements can make up more 2 Miller and Capy than 50%. In genetic model systems like Drosophila melanogaster, in silico analyses have recently indicated that approx 22% of its genome is built up by TEs and their remnants (1). Even in humans, about half of the genome is derived from transposable elements—in particular, long interspersed elements (LINEs), short interspersed elements (SINEs), LTR retrotransposons, and DNA transposons (2). When compared to the genomes of other eukaryotic organisms such as mouse, fly, worm, and mustard weed, the human genome has a much higher density of TEs in the euchromatin. This difference is based on the finding that the vast majority of TEs in humans seem to be more ancient and mainly transpositionally inactive, while in the model organisms mentioned above mobile DNAs are younger and thus still more active (2). TEs are classified into two major groups based on their transposition mecha- nism (3). Class I elements, such as LTR-retrotranposons and LINEs, are char- acterized by DNA sequences with homology to reverse transcriptase, and they are often referred to as retroelements or retrovirus-like elements. Their mobil- ity is achieved through an RNA intermediate that is reverse-transcribed prior to reinsertion, thus mediating a “copy-and-paste” mechanism. This group also includes the SINE elements that use the reverse transcriptase of LINEs. Class II elements are characterized by terminal inverted repeats (TIRs), and they use DNA as a direct-transposition intermediate. They are therefore called DNA transposons and move by a conservative “cut-and-paste” mechanism catalyzed by a transposase. This enzyme is element-encoded in the auto- nomous DNA transposons and is provided in trans for internally deleted, nonautonomous elements. 1.2. Historical Overview In the course of the twentieth century, our vision of the genome dramatically evolved from that of a stable and almost fixed structure to that of a highly flexible and dynamic information storage system. In the first half of the last century, the genome was basically considered as a stable chain of genes located in a head-to-tail organization along chromosomes, slowly evolving by the accumulation of random mutations at constant frequencies. Today such a con- ception is outdated, but it took more than 30 yr to change this dogma (4). Based on her pioneering work on chromosome breakage in maize in the early 1940s, Barbara McClintock provided the first direct experimental evi- dence suggesting that genomes are not static but highly plastic entities (5). Elements involved in these phenomena were initially called “controlling ele- ments.” Based on her observations that some breakage events were always observed at the same chromosomal region, McClintock assumed that these events were due to a particular genetic element named Ds for “Dissociation.” TEs as Natural Molecular Tools 3 In later work she deduced that the instability of Ds elements causing chromo- somal breakage is dependent on the presence of another type of element desig- nated as Ac for “Activator.” Later on in the 1980s, molecular techniques revealed that the Ac–Ds system is composed of autonomous (Ac) and non- autonomous (Ds) copies, whereas only Ac encodes the functional transposase enzyme required for the mobility of both elements (6). Although McClintock’s genetic work was the first clear indication of the existence of mobile DNA elements serving as a major genetic source for genome plasticity, it took more than 30 years before her concept of a dynamic genome became generally accepted (7–9). Between the 1960s and 1970s the following observations paved the way for the discovery and the molecular characterization of mobile DNAs in prokary- otes (reviewed in ref. 4): The discovery of the bacteriophage Mu (10); and the elucidation of IS elements (11–13) as causative agents of mutations, along with their capacity for transmitting antibiotic resistance (14,15). As soon as appro- priate molecular tools were developed for eukaryotic systems in the early 1980s, TEs were recognized immediately as universal components of all living organisms. Two types of theories have been suggested to explain the ubiquitous pres- ence of TEs as well as their high genomic proportions. Soon after the initial discoveries regarding TEs, researchers influenced by the “phenotypic para- digm” of the neo-Darwinian theory broadly speculated that mobile DNAs pro- vide a direct selective advantage to their host organisms. Alternatively, in the light of the emergence of the neutral theory at the end of the 1970s and early 1980s, mobile DNAs were classified as “selfish DNAs” or “ultimate parasites” (16,17). The authors of both classic papers pointed out that the presence and spread of mobile DNAs could be explained solely by their ability to over- replicate the genes of the host genome without invoking a positive selection advantage at the level of the individual organism. As dogmatically stated by Dawkins (18), mobile DNAs are “…genes or genetic material which spread by forming additional copies of itself within the host genome and do not contrib- ute to the phenotype. …” During the last two decades, detailed molecular analyses of transposable elements, focusing on their dynamics and evolution within the host genomes, have modified our perception. Although it is generally accepted at present that mobile DNAs can be regarded as genomic parasites producing mainly neutral and deleterious effects, some of their induced mutations and genomic changes have made significant contributions to the evolution of their hosts (19–21). In this respect these elements can be regarded as a useful genetic load or even as useful parasites (22). 4 Miller and Capy Today, it seems increasingly obvious that genomes can profit from the pres- ence and action of mobile DNAs at various levels bringing about acceleration of genome evolution, as will be detailed in the sections that follow. Of course, mobile DNAs are not the only factor driving genome evolution, but it seems that they could be present at the origin of important events. Therefore, mobile DNAs can be regarded as evolution accelerators, particularly when genomes are facing population and/or environmental stresses (23). In general, TEs are found in all kinds of genomic compartments, such as pericentromeric heterochromatin, telomeres, regulatory regions, exons, and introns. A priori, they can move everywhere in a genome, because their actual genomic target sites consist of a few base pairs only. However, they are not randomly distributed since they are frequently observed in heterochromatin and in regulatory regions. It remains difficult to demonstrate whether they pref- erentially target such regions by target sequence specificity or chromatin accessibility, or instead integrate randomly in the genome with natural selection then retaining and accumulating insertions at particular genomic compartments. In the following sections, we discuss several aspects of the dynamics and evolution of TEs and their interactions with the host genome. Extensive reviews have been published recently covering in detail the broad spectrum of TE–host interactions and their evolutionary consequences (9,19–21). Thus we will first review briefly some of the most important impacts of TEs acting as natural tools on host genome evolution, so that we may then introduce their technical applications as molecular tools and molecular marker systems in modern biology. 2. The Role of TEs as Natural Tools for Shaping Genome Evolution 2.1. Heterochromatin: Only a Wasteland for Transposable Elements? The evolutionary relationship between TEs and heterochromatin is still con- troversial. In general, TEs and their derivatives are found as highly enriched clusters in genomic regions close to the centromere and telomere, and along the chromosomal arms within the intercalary heterochromatin. Obviously TE insertions in heterochromatin are less deleterious than euchromatic insertions, and their concentration in these regions of low gene density might be mainly due to selection against ectopic recombination (24). Indeed, theoretical models have implied that TEs should accumulate in regions with low rates of recombi- nation, such as in the heterochromatin (25,26). Recent experimental data obtained from Drosophila, however, have provided no sufficient support for the hypothesis that the primary reason for the accumulation of mobile DNAs in the heterochromatin is selection against TEs in the euchromatin (27,28). As suggested by Dimitri and Junakovic, “Their accumulation in heterochroma- tin does not seem to be related to intrinsic properties of transposon families … [but could be] determined by some sort of interaction between each transposon TEs as Natural Molecular Tools 5 family and the host genome” (29). The authors conclude that the heterochro- matin might attract de novo insertions of mobile elements mediated by host factors that provide a safe haven to the elements themselves, and thus mini- mize their mutagenic effects in the euchromatin. Moreover, there is also accumulating evidence for direct contribution of TEs in the evolution of heterochromatin. Tandem arrays of engineered P elements inserted in euchromatic positions are sufficient to cause de novo formation of heterochromatin-like structures (30,31), whereas 5S genes do not. Thus, for- mation of heterochromatin seems to have some sort of sequence requirement that is met by at least some sorts of TEs. Although the nature of these proposed special requirements is still unknown, it seems likely that only their structural repetitions are important, thus serving only some structural roles for modify- ing chromatin. Indeed, in Zea mays the Huck retrotransposon seems to provide a structural component the centromeric regions (32–34). Consistent with this conclusion, for example, is the massive insertion of TRIM and TRAM retroelements that has been correlated with heterochromatini- zation of the neo-Y chromosome of D. miranda (35); another example is provided by the functional transition of a formerly active SGM transposon into the structural repetition unit of the main heterochromatic satellite of D. guanche (36). 2.2. TEs and Their Role in Restructuring Chromosomes Barbara McClintock originally discovered mobile DNAs in Zea mays because of their potential to cause chromosomal mutations such as deletions, translocations, and inversions (5). In Drosophila, TEs can be found at the breakpoints flanking chromosomal inversions in both natural populations and laboratory strains (37,38). The hobo element was reported at the breakpoints of three endemic inversions from Hawaiian populations of D. melanogaster (39). In the laboratory strain of the Hikkone line transformed with an active copy of hobo (HFL1), inversions were detected after 50 generations, some of them similar to endemic ones found in natural populations (40). In addition, rare inversions flanked by P elements at the breakpoint were also observed in natural populations collected in the southeastern U.S. (41). Such phenomena are not restricted to D. melanogaster; similar events have been reported from other Drosophila species such as D. buzatii (42). Moreover, it has been shown that all classes of mobile DNAs are capable of causing chromosomal inversions (43,44). 2.3. Emergence of New Genes or New Functions In general, class I elements are defined as using reverse transcriptase (RT) for their own propagation, but in some cases a specific RT enzyme can be recruited for other purposes, such as trans-mobilization of other TEs and 6 Miller and Capy pseudogene formation. For example, SINE elements do not encode the pro- teins require for their retroposition, but use RT encoded from other elements, i.e., LINEs (45,46). Moreover, L1-encoded RT is able to give rise to retro- processed pseudogenes in humans (47). Most of these retrotransposed host gene sequences will evolve like classical pseudogenes, but in some cases such events can initiate the formation of neogenes, which provide a new function to the host. Indeed, retroposition has been viewed as sowing the “seeds” for the evolution of novel gene function (48). As one example, the presence of the Jingwei neogene is restricted to the closely related species D. teissieri, D. yakuba, and D. santomea, belonging to the melanogaster subgroup, and is absent in all other species of Drosophila. This neogene has been originated by the reverse transcription of a spliced Adh mRNA fused to the exons and introns of the yellow emperor gene (49,50). In primates, the chimerical PMCHL neogenes emerged from the initial reverse transcription of the AROM sequence (51,52). Additional cases supporting the important evolutionary role of retroposition in gene evolution have been recently reviewed in detail (53,54). In contrast to the above-mentioned indirect effects on neogene formation induced by retroposition, even the coding section of mobile DNAs can co-opt new host functions, a mechanism designated as “molecular domestication” (20,55,56). For instance, the non-LTR retrotransposons TART and Het-A are exclusively found at the telomeric positions of Drosophila chromosomes (57– 59). Because Drosophila lacks conventional telomeres and telomerase, these retroelements play an essential role in counteracting the erosion of chromo- somal ends and thus providing a substitute for telomerase function to the host. Molecular domestication of mobile DNAs is not restricted to class I ele- ments. As deduced from the initial sequence analyses of the human genome, at least 45 human host genes with currently unknown function unequivocally stem from the coding region of formerly active class II elements (2). So-called transposase-derived neogenes were earlier isolated from various Drosophila species belonging to the obscura and montium species group (55,56,60). In this case, P element-related neogenes have evolved at least two times inde- pendently from coding derivatives of once-mobile P element transposons in separate lineages of Drosophila. Although the functional properties of the P element-derived neogenes are still unknown in their respective hosts, this sys- tem provides the first case for a multiple independent acquisition of the same type of TE-derived coding section during Drosophila evolution (56). More- over, both independent cases of P element domestication were accompanied by further TE-induced events giving rise to (1) the formation of novel cis- regulatory section by multiple insertions of non-P element-related TEs in the TEs as Natural Molecular Tools 7 obscura group (36) and (2) the de novo synthesis of a new intron by the noncoding sections of the P element in the montium subgroup (60). The most spectacular example of molecular domestication of TEs is the recent finding that a key function of the vertebrate immune system most likely evolved directly from a formerly active DNA transposon approximately 100 mya (61–63). The recombination of the V(D)J locus is catalyzed by two enzymes, RAG1 and RAG2, with significant functional and structural similarities to Tc1 transposons. Furthermore, the binding sites for the major centromere-binding protein (CENP-B) of mammals, the “CENP-B box,” have been shown to match the terminal inverted repeats of the pogo DNA transposon (reviewed in ref. 64), and the protein CENP-B itself is an ancient descendant of a pogo-like transposase with a well-conserved DNA-binding domain (65). These data strongly imply that derivatives of once-mobile DNAs can play important roles in the evolution of essential hosts’ cellular functions, such as telomere elonga- tion, immune response, and chromosome segregation. 2.4. Transposable Elements Are the Wild Cards of the Genome Under stable or slightly variable genomic and ecological conditions, the transposition rate of TEs seems to be relatively low. In natural populations of D. simulans the transposition rate of 412 retrotransposons ranges between 10 –3 to 2 × 10 –3 independent of the copy number in their respective genomes (66). These values are significantly higher than earlier estimations (10 –5 to 10 –3 ), which were mainly deduced from laboratory strains (66–68). Therefore, the transposition rates in laboratory strains seem to be one or two orders of magni- tude lower than those derived from natural populations. As suggested by McClintock as early as the late 1970s, genome restructur- ing mediated by TE activity can be seen as an essential component of the hosts’ response to stress, facilitating the adaptation of populations and species facing changing environments (69). Following this assumption, three essential condi- tions must be fulfilled: (1) TEs have to be capable of responding to stress by enhancing their transcriptional and transpositional activity; (2) the enhanced TE mobility has to be sufficient for generating broad genetic variation within the host genome; and (3), this new genetic variability has to be transmissible from one generation to the next. Several lines of arguments are in agreement with the first criterion. Tran- scription of the Tnt1 retrotransposon of Nicotiana tabacum, for instance, seems to be inducible by several biotic and abiotic stress factors (70–72), followed by an actual enhanced mobility of the retrotransposon (73). Moreover IS elements in bacteria may also play an important role in adaptive mutagenesis (74,75). Significant differences of transposition rates are detectible between natural populations within a given species of Drosophila. Some of these differences 8 Miller and Capy are structured according to the geographical origin of the populations. For instance, the activity of mariner and 412 elements exerts a latitudinal variation pattern along an African–European axis. Whereas mariner shows lati- tudinal variations of the somatic excision rate (76), 412 varies with respect to its copy number (77). Furthermore, developmental temperature (76,78–80) and exposure to insecticides seem to increase the somatic excision rate of mariner from a reporter gene (Meusnier, Guichou, and Capy, unpublished results). Fewer experimental data are available to in order to support the second and third criteria. Mackay studied hybrid dysgenesis in D. melanogaster, finding that it was induced by bursts of P element transpositions (81). In the progeny of dysgenic crosses, the response to selection, i.e., to increase or decrease the abdominal bristle number, is higher than in progeny of nondysgenic parents, suggesting that the mutational activity of the P element is sufficient for caus- ing genetic variability on which selection can operate. Based on this pioneer- ing work, several groups have shown that a number of other traits can be affected by transpositional activity (82–86). Although the concept of stress response seems conclusive, some problems still remain to be solved. First, not all types of TEs might be capable of activating transposition due to stress. This specificity probably results from particular small nucleotide motifs located within the regulatory section of the TE. Indeed, such binding site motifs, similar to the plant defense-response elements, were detected in the Tnt1 element (71). Within the untranslated leader region of the Drosophila copia element, sequence motifs were found similar to the core sequence of the SV40 enhancer (87). Therefore, the potential of a specific TE to respond to spe- cific stress might be caused by the presence and accumulation of specific induc- ible enhancers in their regulatory regions. As stated by McDonald et al. (87): “inter-element selection may favor the evolution of more active enhancers within permissive genetic backgrounds. We propose that LTR retroelements and per- haps other retrotransposons constitute drive mechanisms for the evolution of eukaryotic enhancers which can be subsequently distributed throughout host genomes to play a role in regulatory evolution.” The fact remains that most of the reported cases of stress-induced TE mobi- lizations were assayed in somatic tissues only. However, a long-term adapta- tion of the host to environmental changes requires germline modifications (23). In Drosophila, it was assumed that a product derived from the activity of an element might be transferred to the next generation via the egg cytoplasm, causing maternal effects and in some case even grand-maternal effects (88–92). 3. The Taming of TEs and Their Technical Applications At present a deep and detailed understanding of the complex biology of mobile DNAs and their short-term as well as long-term evolutionary fate and TEs as Natural Molecular Tools 9 consequences within genomes is essential for their successful technical application. Based on their exceptional biological features, TEs provide a valu- able collection of molecular tools and experimental strategies appropriate for elucidating a diverse spectrum of biological questions. The most prominent features of TEs are obviously their invasiveness, the structural and functional consequences caused by their genomic insertions, and their potential ability to cross species boundaries. Therefore, TE-based experi- mental strategies serve as standard key molecular tools in modern biology for investigating the structure, organization, and function of genes and genomes. However, prior to the successful application of a given TE as a mutagenic agent, a marker system, or a genetic vehicle for transgenesis, a detailed analy- sis of the structure, function, and dynamics of the mobile element itself is essential. In this respect several protocols for studying the biology of mobile elements by in vivo, in vitro, or in silico approaches are presented in detail in Chapters 2–7 of this book, ranging from high-resolution detection approaches such as in situ hybridization and Southern blot techniques to biochemical and computational in silico whole-genome analyses. In the rest of this book, a large spectrum of technical applications is provided, including protocols for inser- tional mutagenesis, gene tagging, gene silencing, molecular marker analyses, and genetic transformation systems in arthropods and vertebrates. Transposable elements were initially discovered because of their ability to disrupt genes spontaneously, thus acting as natural mutagenes. In the early 1980s the transposon tagging technique was developed in Drosophila as a strat- egy to clone genes, representing the very first transposon-based, genome-wide approach to study gene function in eukaryotes. In later research the systematic extension of this P element-induced, gene disruption technique finally resulted in a compendium of thousands of P insertion lines, covering one-fourth of the vital genes of D. melanogaster (93). Similar genome-wide, TE-based gene dis- ruption strategies were successfully designed and established for a number of other genetic model systems, ranging from Saccharomyces cerevisiae to mouse. TE-based insertional mutagenesis systems can be applied both to localize and isolate a gene involved in a known function, and to infer the function of a gene known only from its sequence. Finally, the objective is to target a TE into a specific gene of interest for analyses of loss or even gain of function. For a long time the technical ability to target DNA sequences to a specific locus were restricted to genetic systems such S. cerevisiae and mouse, but not avail- able for Drosophila. Currently, Drosophila biologists can choose between two different methods for gene targeting, both utilizing the natural tendency of the cell to repair DNA double-strand breaks left behind after the excision of a DNA transposon. The first method, named the “gene conversion technique,” depends on the presence of a P element insertion close to the gene of interest [...]... A The discovery and significance of mobile genetic elements In Mobile Genetic Elements (Sherrat, D J., ed.) IRL Press, Oxford, 1995, pp 1–17 5 McClintock, B The Discovery and Characterization of Transposable Elements: The Collected Papers of B McClintock Garland, New York, 1987 6 Fedoroff, N., Wessler, S., and Shure, M (1983) Isolation of the transposable maize controlling element Ac and Ds Cell 35,... 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J., and Miller, W J (1997) LTR retrotransposons and the evolution of eukaryotic enhancers Genetica 100, 3–13 88 Bryan, G J and Hartl, D L (1988) Maternally inherited transposons excision in Drosophila simulans Science 240, 215–217 89 Bucheton, A (1979) Non-Mendelian female sterility in Drosophila melanogaster: influence of aging and thermic treatments III Cumulative effects induced by these factors Genetics... screening In the course of their long-term coexistence with mobile elements, host genomes might have evolved mechanisms counteracting the mobility and mutability of TEs A growing body of research suggests that epigenetic regulatory mechanisms such as methylation, heterochromatization, and cosuppression arose originally as defense mechanisms against mobile DNAs (97,98) These findings opened for discussion . Capy Mobile Genetic Elements Protocols and Genomic Applications Volume 260 METHODS IN MOLECULAR BIOLOGY TM METHODS IN MOLECULAR BIOLOGY TM Edited by Wolfgang J. Miller Pierre Capy Mobile Genetic Elements Protocols. Genetic Elements Protocols and Genomic Applications TEs as Natural Molecular Tools 1 1 From: Methods in Molecular Biology, vol. 260: Mobile Genetic Elements Edited by: W. J. Miller and P. Capy ©. effects and in some case even grand-maternal effects (88–92). 3. The Taming of TEs and Their Technical Applications At present a deep and detailed understanding of the complex biology of mobile